Note: Descriptions are shown in the official language in which they were submitted.
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1
WATER-SOLUBLE MESOPORPHYRIN COMPOUNDS
AND METHODS OF PREPARATION
Technical Field
The present invention generally relates to water-soluble
mesoporphyrin compounds and processes for their preparation.
More specifically, one or more embodiments of the invention
relate to processes for making novel pharmaceutical
compositions containing such compounds and use of said
compositions in the treatment of various conditions such as
neonatal and other forms of hyperbilirubinemia.
Background Art
Tin (IV) mesoporphyrin IX chloride or stannsoporfin is a
mesoporphyrin chemical compound having the structure
indicated in Figure 1. It has been proposed for use, for
example, as medicament in the treatment of various diseases
including, for example., psoriasis (U.S. Patent No. 4,782,049
to Kappas et al.) and infant jaundice (for example, in U.S.
Patent Nos. 4,684,637, 4,657,902 and 4,692,440).
Stannsoporfin is also known to inhibit heme metabolism in
mammals, to control the rate of tryptophan metabolism in
mammals, and to increase the rate at which heme is excreted
by mammals (U.S. Patent Nos. 4,657,902 and 4,692,440 both to
Kappas et al.).
Processes for obtaining stannsoporfin are known in the
art. Protoporphyrin IX iron (III) chloride or hemin, of the
structural formula indicated in Figure 2, is commonly used as
starting material. The hemin is generally hydrogenated to
form an intermediate mesoporphyrin IX dihydrochloride, which
is subsequently subjected to tin insertion, yielding
stannsoporfin.
The above-referenced methods for the preparation of the
stannsoporfin, or tin (IV) mesoporphyrin IX, however, result
in a non-water soluble compound. Non-water soluble compounds
are difficult to use as therapeutic agents, absent special
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t. n ..uu u.... .u.. .,... .....
2
delivery modes, such as encapsulation into a tablet or
capsule or via use as a powder. Applications of
stannsoporfin in therapeutic treatment of conditions
affecting neonates, children, and adults have thus been
hindered.
Summary of The Invention
One or more embodiments of the present invention provide
novel methods for the preparation of water-soluble
mesoporphyrin compounds. Specific embodiments provide novel
methods for preparing water soluble metal mesoporphyrin
compounds. Other embodiments of the present invention
provide a novel pharmaceutical composition incorporating a
water-soluble tin mesoporphyrin for use in the treatment of
various ailments, including neonatal hyperbilirubinemia.
According to one or more embodiments, reaction of tin
mesoporphyrin IX dichloride or stannsoporfin with an amino
acid in a basic solution forms a novel final compound, a tin
mesoporphyrin IX amino acid, such as tin (IV) mesoporphyrin
IX arginate. According to one or more embodiments, the final
compound can be frozen and vacuum dried so that it can be
isolated in a substantially pure, water-soluble, solid form
or powder. In one or more embodiments, the substantially
pure water-soluble, solid form or powder can be used via
injection, orally or by transdermal delivery, such as a
transdermal patch, to permit therapeutically useful and
active dose volumes.
Brief Description of The Drawings
Figure 1 illustrates the chemical structure of tin
mesoporphyrin chloride (tin (IV) mesoporphyrin IX dichloride)
or stannsoporfin;
Figure 2 illustrates the chemical structure of
protoporphyrin IX iron (III) chloride or hemin;
Figure 3 illustrates the conversion of protoporphyrin IX
iron (III) chloride (ferriporphyrin chloride or hemin) to
mesoporphyrin IX formate; and
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Figure 4 illustrates the conversion of mesoporphyrin IX formate
to tin mesoporphyrin chloride (tin (IV) mesoporphyrin IX dichloride) or
stannsoporfin.
Best Mode of Carrying Out Invention
It is to be appreciated that the various process parameters
described herein (by way of example only, temperature, time, and
pressure) are approximations and may be varied, and certain steps
may be performed in different order. Before describing several
exemplary embodiments of the invention, it is to be understood
that the invention is not limited to the details of construction
or process steps set forth in the following description. The
invention is capable of other embodiments and of being practiced
or carried out in various ways.
In overview, according to one or more embodiments, a tin
mesoporphyrin compound is reacted with one or more amino acids in
a solution such as a basic solution to produce water-soluble
amino-acid complexes of tin mesoporphyrin or stannsoporfin.
According to one or more embodiments, tin (IV) mesoporphyrin IX
(or stannsoporfin) can be obtained according to a variety of
methods, for example, through the methods disclosed in United
States Patent Number 6,818,763. However, it will be understood
that other methods can be used to produce mesoporphyrin halides
such as tin mesoporphyrin IX dichloride, and the present
invention is not limited to a particular method of mesoporphyrin
production.
According to one or more embodiments, a two-stage
hydrogenation process is used to prepare tin mesoporhyrin. In the
first stage, a reaction mixture of hemin and a hydrogenation catalyst are
subjected to a first elevated temperature for a first period of time. In
certain embodiments, the first stage temperature can be in the range
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of about 85-95 C and the period of time is at least about one
hour, for example, between about 1-3 hours.
In a second stage of hydrogenation, the reaction mixture
is cooled to a second temperature for a second period of
time. For example, the second temperature can be in a range
of about 45-50 C and hydrogenated for a second period of time
of about 3-6 hours, in order to convert substantially all
hemin (protoporphyrin IX iron (III) chloride) to
mesoporphyrin IX formate. In certain embodiments, this
second stage can also be conducted in the presence of formic
acid. The same catalyst may be used as in the first step
described above, so that the two stages of t,he process may be
conducted in the same reactor. Optionally, a further charge
of hydrogen may be supplied to the reactor prior to
commencing the second stage. According to one or more
embodiments, the second hydrogenation stage increases the
yield of the mesoporphyrin IX formate, while reducing the
amount of impurities in the final metal mesoporphyrin halide.
By the method described above, the mesoporphyrin IX
intermediate compound in the present invention is not
isolated as a dihydrochloride, but rather as a formate salt.
It will be understood, of course, that other processes can be
used for the preparation of tin (IV) mesoporphyrin
intermediates.
The mesoporphyrin IX formate may be isolated from a
formic acid solution by the addition of a solvent such as
ether or other organic solvent, leading directly to the
mesoporphyrin IX formate intermediate, which is further
subjected to drying. Ethers such as, for example, methyl
tert-butyl ether, diethyl ether or di-isopropyl ether, among
others, may be used. One specific embodiment of the
invention involves the use of methyl tert-butyl ether.
According to the process described above, less solvent
is required compared to other processes, and such smaller
volumes allow for less filter time to obtain the
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intermediate. In exemplary embodiments, ratios of the amount
of hemin to the amount of solvent of about 1:10 to about 1:20
may be used. In addition, the filtration and washings of the
mesoporphyrin IX formate are rapid. After drying, a crude
5 intermediate formate is obtained, in high yields (about
80-95%) and its purity, established by HPLC, is about or
above 97%.
The insertion of metal into mesoporphyrin IX formate to
obtain metal mesoporphyrin halide is described below with
specific reference to tin, to prepare stannsoporfin.
According to an embodiment of the invention, the
insertion of tin into mesoporphyrin IX formate is illustrated
in Figure 4. In one or more embodiments, mesoporphyrin IX
formate is subjected to heating with a tin (II) carrier in an
acid such as acetic acid, buffered with an acetate ion, in
the presence of an oxidant, at reflux. Tin (II) carriers
such as tin (II) halides or tin (II) acetate can be used.
Suitable acetate counter ions include ammonium, sodium or
potassium ions. Oxidants such as oxygen from air or in pure
form as well as hydrogen peroxide can also be used. In one
exemplary embodiment of the invention, the insertion of metal
into mesoporphyrin IX formate occurs. In one or more
embodiments, mesoporphyrin IX formate is subjected to heating
with tin (II) chloride in acetic acid, buffered with ammonium
acetate, and the reaction is conducted in the presence of
air, at reflux. According to this embodiment, tin
mesoporphyrin dichloride is isolated from the reaction
mixture by the addition of water, followed by filtration to
provide a filter cake. Still according to the exemplary
embodiment, prior to drying at about 90-100 C, the filter
cake is triturated into hot, dilute hydrochloric acid, for
example, at a concentration of about 0.1 N-6N, at about
90-100 C. According to this embodiment, the crude,
substantially pure tin mesoporphyrin chloride (crude tin (IV)
mesoporphyrin IX dichloride) is obtained with a yield of
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6
about 75-95% and a purity of about 95%, as judged by HPLC
analysis.
In accordance with at least one embodiment, the tin
mesoporphyrin IX dichloride obtained by the above-described
process may be further purified by dissolving the product in
an aqueous inorganic base solution, for example, dilute
ammonium hydroxide, followed by treatment with charcoal. The
product is then re-precipitated by addition to an acid
solution, such as acetic acid, hydrochloric acid or a mixture
thereof. The above dissolving, charcoal treatment and
re-precipitation steps may be repeated a number of times,
typically about 1-3 times in order to ensure the desired
purity. Prior to drying, the cake is triturated in hot,
dilute hydrochloric acid of a concentration of about 0.1N-6N,
at a temperature of about 90-100 C, in order to remove any
residual ammonium salts. The tin mesoporphyrin chloride
product (tin (IV) mesoporphyrin IX dichloride or
stannsoporfin) is obtained in a yield of about 50-70%, with
an HPLC purity of about or greater than 97%.
The process described above may also be performed to
produce substantially pure or pharmaceutical quality tin
mesoporphyrin chloride (tin (IV) mesoporphyrin IX dichloride
or stannsoporfin) in large scale quantities, such as
quantities exceeding about 0.lkg through and including
multiple kilogram amounts, by slight modifications of the
above procedure, such as increased reaction or drying times
as appropriate based upon the increase in scale of the
starting reactants. Temperature and pressure times likewise
can be modified as needed within the scope of this invention.
The tin mesoporphyrin chloride product (tin (IV)
mesoporphyrin IX dichloride or stannsoporfin) is obtained in
the large scale production process in a yield of about
60-90%, with an HPLC purity of about 97%.
According to one or more embodiments of the present
invention, tin mesoporphyrin, such as the tin (IV)
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7
mesoporphyrin IX obtained as described above, is then reacted
with one or more amino acids in a solution such as a basic
solution to produce water-soluble amino-acid complexes of tin
(IV) mesoporphyrin IX or stannsoporfin. The amino acid
selected may be one or more of the known amino acids,
including but not limited to arginine, glycine, alanine,
leucine, serine, and lysine. The basic solutions may
comprise any common base in aqueous form, including, but not
limited to, sodium hydroxide, trisodium phosphate, an
hydroxide of an alkali metal (Group IIA), an hydroxide of an
alkaline earth metal (Group IIA) or amines such as ethanol
amine or an aqueous solution of one or more of said bases.
The water soluble compounds of the present invention can
be prepared and administered in a wide variety of oral and
parenteral dosage forms. Thus, the compounds of the present
invention can be administered by injection, that is,
intravenously, intramuscularly, intrathecally,
intracutaneously, subcutaneously, intraduodenally, or
intraperitoneally. Also, the compounds of the present
invention can be administered by inhalation, for example,
intranasally. Additionally, the compounds of the present
invention can be administered transdermally. Moreover, the
compounds of the present invention can be administered
rectally, vaginally, or across any mucosal surface, such as
for example the buccal mucosal of the mouth. It will be
obvious to those skilled in the art that the following dosage
forms may comprise as the active component, either a compound
of Figure I or a corresponding pharmaceutically acceptable
salt of a compound of Figures I or II.
According to one or more embodiments of the invention,
the preparation of pharmaceutical compositions can involve
the use of pharmaceutically acceptable carriers, which can be
either solid or liquid. Solid form preparations include
powders, tablets, pills, capsules, cachets, suppositories,
and dispersible granules. A solid carrier can be one or more
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substances which may also act as diluents, flavoring agents,
solubilizers, lubricants, suspending agents, binders,
preservatives, tablet disintegrating agents, an encapsulating
material, or drug delivery agents, such as liposomal
preparations.
In embodiments including powders, the carrier is a
finely divided solid which is in a mixture with the finely
divided active component. In embodiments including tablets,
the active component is mixed with the carrier having the
necessary binding properties in suitable proportions and
compacted in the shape and size desired.
The powders and tablets preferably contain from about
0.1 to about 50 percent of the active compound. Suitable
carriers include, but are not limited to, magnesium
carbonate, magnesium stearate, talc, sugar, lactose, pectin,
dextrin, starch, gelatin, tragacanth, methyl cellulose,
sodium carboxymethyl cellulose, a low melting wax, cocoa
butter, and the like. The term "preparation" is intended to
include the formulation of the active compound with
encapsulating material as a carrier providing a capsule in
which the active component, with or without other carriers,
is surrounded by a carrier, which is thus in association with
it. Similarly, cachets and lozenges are included. Tablets,
powders, capsules, pills, cachets, and lozenges can be used
as solid dosage forms suitable for oral administration.
For preparing suppositories, a low melting wax, such as
a mixture of fatty acid glycerides or cocoa butter, is first
melted and the active component is dispersed homogeneously
therein, as by stirring. The molten homogeneous mixture is
then poured into convenient sized molds, allowed to cool, and
thereby to solidify.
Liquid form preparations include solutions, suspensions,
and emulsions, for example, water or water propylene glycol
solutions. For parenteral injection, liquid preparations can
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be formulated in solution in aqueous polyethylene glycol
solution.
Aqueous solutions suitable for oral use can be prepared
by dissolving the active component in water and adding
suitable colorants, flavors, stabilizing, and thickening
agents as desired. Aqueous suspensions suitable for oral use
can be made by dispersing the finely divided active component
in water with viscous material, such as natural or synthetic
gums, resins, methyl cellulose, sodium carboxymethyl
cellulose, and other well-known suspending agents.
One or more embodiments of the invention include solid
form preparations which are intended to be converted, shortly
before use, to liquid form preparations for oral
administration. Such liquid forms include solutions,
suspensions, and emulsions. These preparations may contain,
in addition to the active component, colorants, flavors,
stabilizers, buffers, artificial and natural sweeteners,
dispersants, thickeners, solubilizing agents, and the like.
The pharmaceutical preparation is preferably in unit
dosage form. In such form, the preparation is subdivided
into unit doses containing appropriate quantities of the
active component. The unit dosage form can be a packaged
preparation, the package containing discrete quantities of
preparation, such as packeted tablets, capsules, and powders
in vials or ampules. Also, the unit dosage form can be a
capsule, tablet, cachet, or lozenge itself, or it can be the
appropriate number of any of these in packaged form.
The quantity of active component in a unit dose
preparation may be varied or adjusted from about 0.1 to about
50 mg, preferably 0.1 to about 10 mg according to the
particular application and the potency of the active
component and size of the patient. The composition can, if
desired, also contain other compatible therapeutic agents.
According to one or more embodiments, in therapeutic use
as agents for treating neonatal hyperbilirubinemia, the
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compounds utilized in the pharmaceutical methods of this
invention are administered at the initial dosage of about 0.1
mg to about 20 mg per kilogram body weight (IM) daily.
Specific exemplary embodiments involve the use of about 0.5
5 mg to about 5 mg per kilogram body weight (IM) for the
treatment of neonatal hyperbilirubinemia. The dosages,
however, may be varied depending upon the requirements of the
patient, the severity of the condition being treated and the
compound being employed. Determination of the proper dosage
10 for a particular situation is within the skill of the art.
In one embodiment, generally, treatment is initiated with
smaller dosages which are less than the optimum dose of the
compound. Thereafter, the dosage is increased by small
increments until the optimum effect under the circumstance is
reached.
Exemplary embodiments of the invention will be further
described for illustrative purposes with reference to the
following non-limiting examples.
Example 1 - Preparation of Tin (IV) Mesoporphyrin IX
Arginate salt
A) - Preparation of mesoporphyrin IX formate - A 2000 ml
hydrogenation vessel was charged with 40.0 g hemin, 4.0 g 5%
Pd/C (50% water by weight), and 800 ml 96% formic acid.
Since hemin and mesoporphyrin IX formate as well as all
reaction intermediates are reportedly light sensitive
materials, care was taken throughout this entire procedure to
minimize the exposure of the reaction to visible or
ultraviolet light.
The vessel was flushed with a nitrogen flow for 10
minutes. With vigorous stirring, it was then pressurized to
50 psi with hydrogen for ten minutes, depressurized, and the
cycle repeated. The vessel was further pressurized to 50 psi
with hydrogen and the temperature was raised to 90 C over
approximately 20 minutes.
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The hydrogenation reaction was maintained at 90 C and 45-55 psi
for 1-1. 5 hours. The reaction mixture was not stable for extended
periods of time at 90 C. The time at this temperature was
sufficient to dissolve all hemin and convert the majority of this
material to the intermediate and final product, mesoporphyrin IX
formate. The reaction was cooled to 50 C/50 psi over 20 minutes.
The pressure and temperature were maintained for 3 hours. The
reaction mixture was shown to be stable at this temperature for up
to 18 hours. The reaction was cooled to 20-25 C, de-pressurized,
and flushed with nitrogen.
The catalyst was removed by filtration through a bed of 20 g
Celite to produce a filter cake. The filter cake was rinsed with
3X50 ml formic acid, and the filtrate including formic acid and
the filter cake was charged to a 2000 ml three-necked, round-
bottom flask equipped with a magnetic stir bar, thermometer, and
distillation bridge. The formic acid solvent was distilled off
under aspirator vacuum to a residual volume of 200 ml. The
distillation bridge was replaced with an addition funnel. With
moderate agitation, 800 ml methyl tert-butyl ether was added drop
wise over 30-60 minutes. The resultant suspension was agitated at
20-25 C for 60 minutes prior to cooling to-20 to-25 C for 1 to 2
hours. The suspension was filtered under reduced pressure. The
filter cake was rinsed with 100 ml filtrate, followed by 2x50 ml
methyl tert-butyl ether and dried under high vacuum at 40-60 C for
24 hours. About 30-38 g of mesoporphyrin IX formate were obtained
(yield of 75-950).
B)- Preparation of Substantially Pure Tin Mesoporphyrin
Chloride (tin (IV) mesoporphyrin IX dichloride or stannsoporfin).
A dark 1000 ml three-necked, round-bottom flask equipped with a
mechanical stirrer, condenser, bubbler, and an aeration tube was charged
with 30.0 g mesoporphyrin IX formate, 34.5 g tin (II) chloride, 7. 1 g
ammonium acetate, and-600 ml acetic acid. The suspension was stirred at
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20-25 C for 30 minutes. Mesoporphyrin IX formate and tin
mesoporphyrin as well as all reaction intermediates are
reportedly light sensitive materials therefore care was taken
throughout this entire procedure to minimize the exposure of
the reaction to light.
The reaction was warmed to reflux, with aeration, for 3
to 4 hours. The reaction was shown to be stable at 110-115 C
for up to 48 hours. Once complete, the reaction mixture was
cooled to 60-70 C and 300 ml water was added while cooling to
20-25 C over 60 minutes. The suspension was filtered under
reduced pressure. The filter cake was rinsed with 2x60 ml
water. A dark, 1000 ml, three-neck, round-bottom, flask
equipped with a stir bar, thermometer, condenser, and
nitrogen purge was charged with the wet cake from the above
step, and 500 ml 1 N HC1. The resultant suspension was
warmed to 90C for 1 hour. The suspension was filtered under
reduced pressure. The filter cake was rinsed with 2x50 ml
0.1N HC1 and dried under high vacuum at 80-90 C for 24 hours.
About 25 to 28 g of crude, substantially pure (about or
exceeding 95% purity) tin mesoporphyrin chloride (tin (IV)
mesoporphyrin IX dichloride or stannsoporfin) was obtained
for a yield of about 83-93%.
C) - Preparation of the Arginate Salt. The tin (IV)
mesoporphyrin IX dichloride prepared according to the above
referenced process is then combined with a solution of
arginine in aqueous sodium hydroxide and mixed for a period
of sufficient time such that the reaction is closer to
completion. The ratio of the tin (IV) mesoporphyrn IX
dichloride to the arginine is about 2:1. The ratio of the tin
mesoporphyrin IX dichloride to the aqueous sodium hydroxide
is about 1:3. The solution is then filtered, rinsed with
deionized water and frozen. Following freezing of the
filtrate solution including the liquid and the tin
mesoporphyrn-amino acid complex, the frozen solution is
vacuum dried to provide in a lyophilized product.
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= t ....J .n..x .mw . nrv t .x M=n a t
13
Example 2 - Preparation of an Injectible or Transdermal
Formulation of Tin (IV) Mesoporphyrin IX Arginate salt
A) - Preparation of mesoporphyrin IX formate - A 2000 ml
hydrogenation vessel was charged with 40.0 g hemin, 4.0 g 5%
Pd/C (50% water by weight), and 800 ml 96% formic acid.
Since hemin and mesoporphyrin IX formate as well as all
reaction intermediates are reportedly light sensitive
materials, care was taken throughout this entire procedure to
minimize the exposure of the reaction to visible or
ultraviolet light.
The vessel was flushed with a nitrogen flow for 10
minutes. With vigorous stirring, it was then pressurized to
50 psi with hydrogen for ten minutes, depressurized, and the
cycle was repeated. The vessel was further pressurized to 50
psi with hydrogen and the temperature was raised to 90 C over
approximately 20 minutes.
The hydrogenation reaction was maintained at 90 C and
45-55 psi for 1-1.5 hours. The reaction mixture was not
stable for extended periods of time at 90 C. The time at
this temperature was sufficient to dissolve all hemin and
convert the majority of this material to the intermediate and
final product, mesoporphyrin IX formate. The reaction was
cooled to 50 C/50 psi over 20 minutes. The pressure and
temperature were maintained for 3 hours. The reaction
mixture was shown to be stable at this temperature for up to
18 hours. The reaction was cooled to 20-25 C,
de-pressurized, and flushed with nitrogen.
The catalyst was removed by filtration through a bed of
20 g celite. The filter cake was rinsed with 3x50 ml formic
acid and the filtrate was charged to a 2000 ml three-necked,
round-bottom flask equipped with a magnetic stir bar,
thermometer, and distillation bridge. The formic acid
solvent was distilled off under aspirator vacuum to a
residual volume of 200 ml. The distillation bridge was
replaced with an addition funnel. With moderate agitation,
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800 ml methyl tert-butyl ether was added drop wise over 30-60
minutes. The resultant suspension was agitated at 20-25 C
for 60 minutes prior to cooling to -20 to -25 C for 1 to 2
hours. The suspension was filtered under reduced pressure.
The filter cake was rinsed with 100 ml filtrate, followed by
2x50 ml methyl tert-butyl ether and dried under high vacuum
at 40-60 C for 24 hours. About 30-38 g of mesoporphyrin IX
formate was obtained (yield of 75-950).
B) - Preparation of Substantially Pure Tin Mesoporphyrin
Chloride (tin (IV) mesoporphyrin IX dichloride or
stannsoporfin). A dark 1000 ml three necked, round-bottom
flask equipped with a mechanical stirrer, condenser, bubbler,
and an aeration tube was charged with 30.0 g mesoporphyrin IX
formate, 34.5 g tin (II) chloride, 7.1 g ammonium acetate,
and 600 ml acetic acid. The suspension was stirred at
20-25 C for 30 minutes. Mesoporphyrin IX formate and tin
mesoporphyrin as well as all reaction intermediates are
reportedly light sensitive materials therefore care was taken
throughout this entire procedure to minimize the exposure of
the reaction to light.
The reaction was warmed to reflux, with aeration, for 3
to 4 hours. The reaction was shown to be stable at 110-115 C
for up to 48 hours. Once complete, the reaction mixture was
cooled to 60-70 C and 300 ml water were added while cooling
to 20-25 C over 60 minutes. The suspension was filtered
under reduced pressure. The filter cake was rinsed with 2x60
ml water. A dark, 1000 ml, three-neck, round-bottom, flask
equipped with a stir bar, thermometer, condenser, and
nitrogen purge was charged with the wet cake from the above
step, and 500 ml 1N HCI. The resultant suspension was warmed
to 90 C for 1 hour. The suspension was filtered under
reduced pressure. The filter cake was rinsed with 2x50 ml
0.1N HCI and dried under high vacuum at 80-90 C for 24 hours.
About 25 to 28 g of crude, substantially pure (about or
exceeding 95% purity) tin mesoporphyrin chloride (tin (IV)
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mesoporphyrin IX dishloride or stannsoporfin) was obtained for a
yield of about 83-93%.
C)- Preparation of the Arginate Salt. The tin (IV)
mesoporphyrin IX dichloride prepared according to the process
5 described above, is combined with an excess solution of arginine in
aqueous sodium hydroxide (the ratio being about 2: 1: 3) and mixed
for a sufficient period of time as to affect dissolution. The ratio
of the tin (IV) mesoporphyrin IX dichloride to the arginine is about
2: 1. The ratio of the tin mesoporphyrin IX dichloride to the
10 aqueous sodium hydroxide is 1: 3. The solution is then filtered,
rinsed with deionized water and frozen. Following freezing of the
solution, the frozen solution is vacuum dried to result in a
lyophilized product.
We expect that the reconstituted product can be resolubilized
15 into DI H20 or 5% saline, or into one of other known in the art
injectible or transdermal solutions, and delivered to the patient
by such injectible or transdermal methods. Those skilled in the art
would readily appreciate that other amino acids would similarly
react with tin (IV) mesoporphyrin IX dichloride to form a water
soluble complex consistent with this invention.
For example, we have prepared and reacted a number of amino
acids with tin-mesoporphyrin in the presence of NaOH. The material
isolated from these reactions have been examined by 'H NMR and
UV/VIS (ultraviolet and visible) spectroscopy, as well as comparing
the solubilities of the reaction products have also been compared
to the solubilities of their respective starting materials, to
determine whether an amino acid-tin-mesoporphyrin complex forms form
such a reaction.
Comparison of the 'H NMR spectra of the starting materials to
the reaction products essentially reveals a 1: 1 mixture of amino
acid and tin-mesoporphyrin. Since the 'H NMR methodology as
explained in the art indicates that 'H NMR spectroscopy may not be
sensitive enough to detect the
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formation of the desired complexes, we also utilized UV/VIS
spectroscopy as an analytical method. The UV/VIS spectroscopy
revealed small discrete changes in the spectrum that are
likely consistent with the formation of a complex between
amino acid and tin-mesoporphyrin.
Solubility profile changes likewise suggest the
formation of a novel species, the amino acid -
tin-mesoporphyrin complex, upon mixing the tin mesoporhyrin
and an amino acid in the presence of NaOH due to a change in
the solubility of the amino acids component of the reaction
mixture, as indicated in the table below (Methanol= MeOH;
Isopropyl alcohol= IPA; Ethyl Acetate= EtOAc; tetra
hydrofuran= THF; Methy-t-butylether= MTBE:
Compound Water MeOH IPA EtOAc
Tin mesoporphyrin Y Y S N
sodium
L-alanine sodium Y Y N N
L-leucine sodium Y N N N
L-serine sodium Y N N N
L-arginine sodium Y N N N
L-glycine sodium Y N N N
L-alanine-complex Y Y N N
L-leucine complex Y Y N N
L-serine-complex Y Y N N
L-arginine-complex Y Y N N
L-lysine complex Y Y N N
L-glycine-complex Y Y N N
We further reacted a number of additional amino acids
with tin mesoporhyrin in.the presence of NaOH in accordance
with the present invention. The material isolated from these
reactions has been examined by 'H NMR and UV/VIS spectroscopy.
The solubilities of these additional reaction products have
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also been compared to the solubilities of their respective
starting materials.
The amino acids L-histidine, L-phenylalanine and
L-tyrosine are different from the amino acids reacted so far
in that they each contain an aromatic moiety. ATi-Tt
interaction between the respective aromatic moieties of the
porphyrin and said amino acids produces an obvious chemical
shift change in the 'H NMR spectra of the complexes. The 'H
NMR spectra of the complexes exhibit a significant up-field
shift in the tin mesoporphyrin 'H NMR resonances found at -
10.5 ppm. In the complexes, these resonances are found at
9.5 ppm. Examination of the UVIVIS spectra also shows
significant changes in the absorption found at -350 nm.
These absorption changes indicate the likely formation of
chemical bonds between the amino acid and the tin
mesoporphyrin, further evidencing a formation of a complex
between the amino acid and tin mesoporphyrin.
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Starting Materials
Compound Water MeOH IPA EtOAc
Tin Y Y S N
mesoporphyrin sodium
L-alanine Y Y N N
sodium
L-leucine Y N N N
sodium
L-lysine sodium Y N N N
L-glycine Y N N N
sodium
L-histidine Y N N N
L-phenylalanine Y N N N
L-tyrosine S S S N
Complexes
L-alanine-complex Y Y N N N
L-leucine-complex Y Y N N N
L-serine-complex Y Y N N N
L-arginine-complex Y Y N N N
L-lysine-complex Y Y N N N
L-glycine-complex Y Y N N N
L-histidine-complex Y Y N N N
L-phenylalanine-complex Y Y N N N
L-tyrosine-complex Y Y N N N
Finally, we reacted all naturally known amino acids with
tin mesoporphyrin in accord with the present invention. The
amino acids were allowed to react with tin mesoporphyrin in
the presence of NaOH in water. The material isolated from
these reactions were examined by 1H NMR and uV/VIS
spectroscopy. The solubilities of the reaction products were
then also compared to the solubilities of their respective
starting materials.
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Starting Materials
Compound Water MeOH IPA EtOAc
Tin Y Y S N
mesoporphyrin sodium
L-alanine Y Y N N
sodium
L-leucine Y N N N
sodium
L-serine sodium Y N N N
L-arginine Y N N N
sodium
L-lysine sodium Y N N N
L-glycine Y N N N
sodium
L-histidine Y N N N
sodium
L-phenylalanine Y N N N
sodium
L-tyrosine Y S N N
sodium
DL-tryptophan Y Y N N
sodium
L-proline
sodium
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Complexes
L-alanine-complex Y Y N N N
L-leucine-complex Y Y N N N
L-serine-complex Y Y N N N
L-arginine-complex Y Y N N N
L-lysine-complex Y Y N N N
L-glycine-complex Y Y N N N
L-histidine-complex Y Y N N N
L-phenylalanine-complex Y Y N N N
L-tyrosine-complex Y Y N N N
DL-tryptophan-complex Y Y N N N
L-proline-complex
A number of the reaction products exhibit different
solubility properties when compared to the solubility
5 properties of their respective starting materials. As a
result, a change in the solubility of the amino acids
component of the reaction mixture suggests and supports
formation of a tin-mesoporphyrin-amino acid complex.
Accordingly, using the change in solubility properties of the
10 amino acids as a guide, the above table suggests and supports
complex formation between tin mesoporphyrin and all the amino
acids listed. We expect similar complex formation with tin
mesoporphyrin and the amino acid proline based on the other
amino acid results as tabulated.
15 For many of the reaction products, differences ranging
from subtle to obvious were observed between the UV/VIS
spectra of tin mesoporphyrin sodium and the reaction
products. This change is suggestive of the formation of a
new chemical species. For the reaction products derived from
20 the amino acids glycine sodium, alanine sodium, leucine
sodium, lysine sodium and serine sodium, a shoulder appears
on the peak at -400 nm in the UV/VIS spectrum. A more
dramatic change is observed for the reaction products derived
from the amino acids histidine sodium, phenylalanine sodium,
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tyrosine sodium, tryptophan sodium, methionine sodium and
threonine sodium, where an altogether new absorbance is
observed at -380 nm in the UV/VIS spectrum. The UV/VIS
spectrum for the reaction product derived from arginine
sodium remains essentially unchanged when compared to the
UVNIS spectrum of tin mesoporphyrin sodium. Thus, it is
likely that the biochemical and therapeutic properties of
tin-mesoporphyrin would be exhibited in the novel
tin-mesoporphyrin - amino acid complexes formed by the
present invention.
Although the invention herein has been described with
reference to particular embodiments, it is to be understood
that these embodiments are merely illustrative of the
principles and applications of the present invention. It is
therefore to be understood that numerous modifications may be
made to the illustrative embodiments and that other
arrangements may be devised without departing from the spirit
and scope of the present invention as defined by the appended
claims.
